Project Summary Our application entitled ?Mechanisms of mRNA translation that modulate protein aggregation? investigates how mechanisms of translation modulate the propensity of newly synthesized proteins to aggregate. Studies in yeast and mammalian cells have shown that pretreatment of cells with cycloheximide, an inhibitor of protein synthesis, prevents heat shock-induced protein aggregation, revealing a vulnerability of newly synthesized proteins to aggregate. Our mechanistic work investigating how tetracyclic antibiotics like minocycline prevent protein aggregation, revealed that pre-treating cells with minocycline also prevents heat shock-induced protein aggregation. However, in contrast to cycloheximide, which blocks translation completely, minocycline only reduces over-all translation by -25%. Further investigations into the mechanism by which minocycline modulates translation revealed minocycline to bind to the 40S subunit of the ribosome and to reduce `ribosomal load,' defined as the number of ribosomes per mRNA. Consequently, minocycline preferentially reduces translation of highly expressed mRNAs that are translated by heavy polysomes but has very little effect on already lowly expressed mRNAs. Based on these findings, we hypothesize that translation by high density polysomes strains the capacity of the protein folding machinery by synthesizing hundreds of copies of the same protein in a short period of time. This increases the risk of aggregation as hundreds of nascent proteins locally compete for the same folding factors. This risk of aggregation is likely to be intensified during stress or in cases in which biological signals such as inflammatory signals dramatically alter gene expression in a cell, leading to intense translational activity. While young organisms have sufficient folding capacity to absorb a sudden and intense increase in translation, older organisms might not, as the folding capacity has progressively declined with age. Minocycline treatment, by reducing polysome formation, reduces aggregation. In this application we will generate a rich portrait of translation by translational state analysis and ribosome profiling before the induction of a heat shock. Subsequent analysis of the aggregates by proteomics will reveal the identity of the aggregating proteins and allow us to link aspects of translation such as ribosomal load to aggregation. Perturbing translation by exposing the cells to minocycline to reduce ribosomal load, or to TNF? to alter the set of mRNAs experiencing the highest load, will reveal how translation modulates the propensity of newly synthesized proteins to aggregate. The successful completion of these studies will reveal new opportunities to modulate protein aggregation that can be exploited for therapeutic development including the design of eukaryotic tetracyclines retaining their anti-aggregation effects but lacking antibiotic activity.